Target (protein/gene name): N-acetylneuraminic acid synthase NCBI Gene # or RefSeq #: 902174 Protein ID (NP or XP #) or Wolbachia#: NP_273133.1 Organism (including strain):Neisseria meningitidis MC58 Etiologic Risk Group: 2 Disease Information: Meningitis is the inflammation of the meninges, the thin layer of membranes that covers the brain and the spinal cord. There are three types of infections: bacterial, viral, and fungal. The most contagious and lethal form is bacterial meningitis; death can occur in as little as a few hours. In the United States, approximately 4,100 cases of bacterial meningitis, including 500 deaths, occurred annually from 2003-2007. Meningitis symptoms include sudden onset of fever, headache, and stiff ache. Additional symptoms include nausea, vomiting, increased sensitivity to light, and confusion. From 1/3 to 1/2 of the survivors face permanent physical or mental damages. Neisseria meningitidis (a gram-negative, aerobic, non-endospore forming diplococcus) is one of several bacteria responsible for the crippling disease. It can be spread from person to person through coughing and sneezing. Inside the nasal cavities, the bacteria quickly divide to enter the bloodstream. Once the bacteria enter the subarachnoid space, the host’s immune system is unable to suppress the infection from developing in the cerebrospinal fluid because the region lacks the necessary antibodies and white blood cells. Link to TDR Targets page (if present): N/A Link to Gene Database page (NCBI, EuPath databases, -e.g. TryTryp, PlasmoDB, etc –or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/902174 (The site titles the gene as “polysialic acid capsule biosynthesis protein SiaC” which is another name for N-acetylneuraminic acid synthase) Essentiality of the Protein: The pathogens produce sialylated capsular polysaccharides, mimicking the exterior surface of mammalian cells and consequently concealing the bacteria from the host’s immune system Is it a monomer or multimer as biological unit? Dimer Complex of proteins: N/A Druggable target (list number or cite evidence from a paper/database showing draggle in another organism): One potential inhibitor discovered in la http://pubs.acs.org/doi/abs/10.1021/bi9012758. Also, Brenda shows inhibitors for the target in other organisms. EC#: 2.5.1.56 Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.56&Suchword=&organism%5B%5D=Neisseria+meningitidis&show_tm=0 Show screenshot of BRENDA enzyme mechanism schematic
Figure 1: Reaction catalyzed by N-acetylneuraminate synthase. The reaction is the condensation of phosphoenolpyruvate and -acetyl-D-mannosamine to N-acetylneuraminate.
Enzyme Assay Information (spectrophotometric, coupled assay, reagents): Continuous spectrophotometric assay Continuous coupled assay for phosphate Link to Sigma (or other company) page for assay or link (or citation) to paper that contains assay information: http://pubs.acs.org/doi/full/10.1021/bi9012758 http://www.pnas.org/content/89/11/4884.full.pdf List cost & quantity of substrate reagents, suppliers, & Catalogs #:
Figure 2: Structure of NmNAS (PDB: 1XUZ). Both Unit A (tri-colored) and B (gray) shown as cartoon without the side chain. The catalytic, linker, and AFPL domains colored as red, orange, and blue, respectively. ManAC (green) and PEP (black) shown as stick. The essential cofactor Mn2+ shown as pink spheres.
Amino Acid Sequence:
>1XUZ:A|PDBID|CHAIN|SEQUENCE:
MQNNNEFKIGNRSVGYNHEPLIICEIGINHEGSLKTAFEMVDAAYNAGAEVVKHQTHIVEDEMSDEAKQVIPGNADVSIY
EIMERCALNEEDEIKLKEYVESKGMIFISTPFSRAAALRLQRMDIPAYKIGSGECNNYPLIKLVASFGKPIILSTGMNSI
ESIKKSVEIIREAGVPYALLHCTNIYPTPYEDVRLGGMNDLSEAFPDAIIGLSDHTLDNYACLGAVALGGSILERHFTDR
MDRPGPDIVCSMNPDTFKELKQGAHALKLARGGKKDTIIAGEKPTKDFAFASVVADKDIKKGELLSGDNLWVKRPGNGDF
SVNEYETLFGKVAACNIRKGAQIKKTDIE Length of your proteins in Amino Acids: 349 Molecular weight of your proteins in kiloDaltons using the Expasy ProtParam Website: 38390.7kDa Molar Extinction Coefficient of your protein at 280nm wavelength: 21890 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html) Input your amino acid sequence to it
Figure 3: TMPred prediction of transmembrane region. There is one predicted transmembrane region; however, based on N-acetylneuraminate synthase's function in the pathogen, it is very unlikely to be a true transmembrane region.
CDS Gene Sequence: ATGCAAAACAACAACGAATTTAAAATTGGTAATCGTTCAGTAGGTTACAACCACGAACCATTGATTATCT GTGAAATCGGCATCAATCATGAAGGCTCTTTAAAAACAGCTTTTGAAATGGTTGATGCTGCCTATAATGC AGGCGCTGAAGTTGTTAAACATCAAACACACATCGTTGAAGACGAAATGTCTGATGAGGCCAAACAAGTC ATTCCAGGCAATGCAGATGTCTCTATTTATGAAATTATGGAACGTTGCGCCCTGAATGAAGAAGATGAGA TTAAATTAAAAGAATACGTAGAGAGTAAGGGTATGATTTTTATCAGTACTCCTTTCTCTCGTGCAGCTGC TTTACGATTACAACGTATGGATATTCCAGCATATAAAATCGGCTCTGGCGAATGTAATAACTACCCATTA ATTAAACTGGTGGCCTCTTTTGGTAAGCCTATTATTCTCTCTACCGGCATGAATTCTATTGAAAGCATCA AAAAGTCGGTAGAAATTATTCGAGAAGCAGGGGTACCTTATGCTTTGCTTCACTGTACCAACATCTACCC AACCCCTTACGAAGATGTTCGATTGGGTGGTATGAACGATTTATCTGAAGCCTTTCCAGACGCAATCATT GGCCTGTCTGACCATACCTTAGATAACTATGCTTGCTTAGGAGCAGTAGCTTTAGGCGGTTCGATTTTAG AGCGTCACTTTACTGACCGCATGGATCGCCCAGGTCCGGATATTGTATGCTCTATGAATCCGGATACTTT TAAAGAGCTCAAGCAAGGCGCTCATGCTTTAAAATTGGCACGCGGCGGCAAAAAAGACACGATTATCGCG GGAGAAAAGCCAACTAAAGATTTCGCCTTTGCATCTGTCGTAGCAGATAAAGACATTAAAAAAGGAGAAC TGTTGTCCGGAGATAACCTATGGGTTAAACGCCCAGGCAATGGAGACTTCAGCGTCAACGAATATGAAAC ATTATTTGGTAAGGTCGCTGCTTGCAATATTCGCAAAGGTGCTCAAATCAAAAAAACTGATATTGAATAA GC% Content for gene: 40.762% CDS Gene Sequence (codon optimized) copy from output of Primer Design Protocol: ATGGAAAAAGTTAATGAAGAACGTGACGCGGTGTTTGAAGATCACATCGGCGACCGTCGCCGCTCTGTTCGTTCTCTGCTCGAAGAAGCTTTCGCGGACGAAATGGAGAAAACCTCTTACGACGTTGAGGTTGCGGATACCCCACAGCCGCACATCCCAATCCGTTTTCGCCACCCGCCGATCGCTGGCCCAGTTCATGATGTGTTCGGTGATGCTATCCACGACATCTTTCAGAAAATGATGAAACGCGGTCAGGCTGTTGACTTCTGCCACTGGGTGTCTCACCTGATTGCGACTGAAATCGACGAAAAGTTTTCCGAAGTTGCGTTCCGTGACGTTCAGTACAACCCGGACATCTACGTTACGGACTCTACTACCGAAGCGAAAAAACTGTTCAACGATAAGATTTGGCCTGCGATCGATAAAATCCTGCAGCAAAACGCCGAAACCTGTCCGATCCTGTCTGAGAAATGGTCTGGTATTCACGTTAGCGGTGATCAGCTCAAGGGTCAGCGCCACAAACAGGAAGATCGCTTCCTGGCTTACCCTAACGGTCAATATATGGACCGTGGTGAGGACCCGATTTCTGTTCTGGCGGTTTTCGATGGTCACGGCGGTCACGAATGCTCTCAGTACGCGGCAGGTCATCTGTGGGAAACGTGGCTGGAGGTGCGTAAAAGCCGCGACCCGTCTGACTCTCTGGAGGACCAGCTGCGCAAATCTCTCGAACTGCTGGACGAACGTATGACCGTGCGTTCTGTCAAAGAATGCTGGAAAGGCGGTTCTACCGCTGTCTGCTGTGCAATCGACATGGACCAGAAGCTCATGGCGCTCGCATGGCTGGGCGACTCTCCGGGCTATGTTATGTCTAACATCGAATTCCGTCAACTCACTCGTGGTCATTCTCCTTCTGATGAGCGCGAAGCACGTCGCGTAGAGGAGGCAGGTGGCCAACTCTTTGTAATCGGTGGTGAACTCCGTGTTAACGGTGTGCTGAACCTGACGCGCGCACTCGGCGATGTTCCGGGTCGTCCGATGATCTCTAACGAACCGGAAACTTGCCAGGTTCCGATCGAATCTAGCGACTATCTCGTGCTCCTGGCGTGCGATGGTATCAGCGACGTATTCAATGAGCGTGACCTGTATCAACTGGTCGAAGCGTTTGCCAATGATTACCCGGTTGAAGACTACGCGGAACTGTCTCGCTTTATTTGCACCAAGGCCATTGAAGCAGGCTCTGCGGACAATGTATCTGTTGTTATTGGTTTCCTGCGTCCGCCGCAGGACGTGTGGAAACTGATGAAGCATGAATCTGACGACGAAGACTCTGATGTTACCGATGAGGAATAA
GC% Content for gene (codon optimized): 52.59%
Primer design results for 'tail' primers (this is just 2 sequences):
NCBI Gene # or RefSeq #: 902174
Protein ID (NP or XP #) or Wolbachia#: NP_273133.1
Organism (including strain): Neisseria meningitidis MC58
Etiologic Risk Group: 2
Disease Information:
Meningitis is the inflammation of the meninges, the thin layer of membranes that covers the brain and the spinal cord. There are three types of infections: bacterial, viral, and fungal. The most contagious and lethal form is bacterial meningitis; death can occur in as little as a few hours. In the United States, approximately 4,100 cases of bacterial meningitis, including 500 deaths, occurred annually from 2003-2007. Meningitis symptoms include sudden onset of fever, headache, and stiff ache. Additional symptoms include nausea, vomiting, increased sensitivity to light, and confusion. From 1/3 to 1/2 of the survivors face permanent physical or mental damages. Neisseria meningitidis (a gram-negative, aerobic, non-endospore forming diplococcus) is one of several bacteria responsible for the crippling disease. It can be spread from person to person through coughing and sneezing. Inside the nasal cavities, the bacteria quickly divide to enter the bloodstream. Once the bacteria enter the subarachnoid space, the host’s immune system is unable to suppress the infection from developing in the cerebrospinal fluid because the region lacks the necessary antibodies and white blood cells.
Link to TDR Targets page (if present): N/A
Link to Gene Database page (NCBI, EuPath databases, -e.g. TryTryp, PlasmoDB, etc –or PATRIC, etc.) http://www.ncbi.nlm.nih.gov/gene/902174 (The site titles the gene as “polysialic acid capsule biosynthesis protein SiaC” which is another name for N-acetylneuraminic acid synthase)
Essentiality of the Protein:
The pathogens produce sialylated capsular polysaccharides, mimicking the exterior surface of mammalian cells and consequently concealing the bacteria from the host’s immune system
Is it a monomer or multimer as biological unit? Dimer
Complex of proteins: N/A
Druggable target (list number or cite evidence from a paper/database showing draggle in another organism):
One potential inhibitor discovered in la http://pubs.acs.org/doi/abs/10.1021/bi9012758. Also, Brenda shows inhibitors for the target in other organisms.
EC#: 2.5.1.56
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.56&Suchword=&organism%5B%5D=Neisseria+meningitidis&show_tm=0
Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay Information (spectrophotometric, coupled assay, reagents):
Continuous spectrophotometric assay
Continuous coupled assay for phosphate
Link to Sigma (or other company) page for assay or link (or citation) to paper that contains assay information:
http://pubs.acs.org/doi/full/10.1021/bi9012758
http://www.pnas.org/content/89/11/4884.full.pdf
List cost & quantity of substrate reagents, suppliers, & Catalogs #:
Structure (PDB or Homology Model): 1XUU, 1XUZ
Current inhibitors:
- 5-acetamido-4,6,7,8,9-pentahydroxy-2-phosphoryl nonanoic acid triethylammonium salt
Inhibitors for the target in other organisms are found on Brenda .
http://www.brenda-enzymes.org/enzyme.php?ecno=2.5.1.56&Suchword=&organism%5B%5D=Neisseria+meningitidis&show_tm=0
Expression Information (has it been expressed in bacterial cells):
- Has been expressed in E. Coli BL21(DE3) (http://www.sciencedirect.com/science/article/pii/S0006291X05023570) (http://pubs.acs.org/doi/full/10.1021/bi400062c)
- Found in the cytoplasm
Purification Method:
- Ni2+ Chromatography
Image of Protein (PyMol with features delineated & shown separately):
Amino Acid Sequence:
>1XUZ:A|PDBID|CHAIN|SEQUENCE:
MQNNNEFKIGNRSVGYNHEPLIICEIGINHEGSLKTAFEMVDAAYNAGAEVVKHQTHIVEDEMSDEAKQVIPGNADVSIY
EIMERCALNEEDEIKLKEYVESKGMIFISTPFSRAAALRLQRMDIPAYKIGSGECNNYPLIKLVASFGKPIILSTGMNSI
ESIKKSVEIIREAGVPYALLHCTNIYPTPYEDVRLGGMNDLSEAFPDAIIGLSDHTLDNYACLGAVALGGSILERHFTDR
MDRPGPDIVCSMNPDTFKELKQGAHALKLARGGKKDTIIAGEKPTKDFAFASVVADKDIKKGELLSGDNLWVKRPGNGDF
SVNEYETLFGKVAACNIRKGAQIKKTDIE
Length of your proteins in Amino Acids: 349
Molecular weight of your proteins in kiloDaltons using the Expasy ProtParam Website: 38390.7kDa
Molar Extinction Coefficient of your protein at 280nm wavelength: 21890
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html) Input your amino acid sequence to it
CDS Gene Sequence:
ATGCAAAACAACAACGAATTTAAAATTGGTAATCGTTCAGTAGGTTACAACCACGAACCATTGATTATCT
GTGAAATCGGCATCAATCATGAAGGCTCTTTAAAAACAGCTTTTGAAATGGTTGATGCTGCCTATAATGC
AGGCGCTGAAGTTGTTAAACATCAAACACACATCGTTGAAGACGAAATGTCTGATGAGGCCAAACAAGTC
ATTCCAGGCAATGCAGATGTCTCTATTTATGAAATTATGGAACGTTGCGCCCTGAATGAAGAAGATGAGA
TTAAATTAAAAGAATACGTAGAGAGTAAGGGTATGATTTTTATCAGTACTCCTTTCTCTCGTGCAGCTGC
TTTACGATTACAACGTATGGATATTCCAGCATATAAAATCGGCTCTGGCGAATGTAATAACTACCCATTA
ATTAAACTGGTGGCCTCTTTTGGTAAGCCTATTATTCTCTCTACCGGCATGAATTCTATTGAAAGCATCA
AAAAGTCGGTAGAAATTATTCGAGAAGCAGGGGTACCTTATGCTTTGCTTCACTGTACCAACATCTACCC
AACCCCTTACGAAGATGTTCGATTGGGTGGTATGAACGATTTATCTGAAGCCTTTCCAGACGCAATCATT
GGCCTGTCTGACCATACCTTAGATAACTATGCTTGCTTAGGAGCAGTAGCTTTAGGCGGTTCGATTTTAG
AGCGTCACTTTACTGACCGCATGGATCGCCCAGGTCCGGATATTGTATGCTCTATGAATCCGGATACTTT
TAAAGAGCTCAAGCAAGGCGCTCATGCTTTAAAATTGGCACGCGGCGGCAAAAAAGACACGATTATCGCG
GGAGAAAAGCCAACTAAAGATTTCGCCTTTGCATCTGTCGTAGCAGATAAAGACATTAAAAAAGGAGAAC
TGTTGTCCGGAGATAACCTATGGGTTAAACGCCCAGGCAATGGAGACTTCAGCGTCAACGAATATGAAAC
ATTATTTGGTAAGGTCGCTGCTTGCAATATTCGCAAAGGTGCTCAAATCAAAAAAACTGATATTGAATAA
GC% Content for gene: 40.762%
CDS Gene Sequence (codon optimized) copy from output of Primer Design Protocol:
ATGGAAAAAGTTAATGAAGAACGTGACGCGGTGTTTGAAGATCACATCGGCGACCGTCGCCGCTCTGTTCGTTCTCTGCTCGAAGAAGCTTTCGCGGACGAAATGGAGAAAACCTCTTACGACGTTGAGGTTGCGGATACCCCACAGCCGCACATCCCAATCCGTTTTCGCCACCCGCCGATCGCTGGCCCAGTTCATGATGTGTTCGGTGATGCTATCCACGACATCTTTCAGAAAATGATGAAACGCGGTCAGGCTGTTGACTTCTGCCACTGGGTGTCTCACCTGATTGCGACTGAAATCGACGAAAAGTTTTCCGAAGTTGCGTTCCGTGACGTTCAGTACAACCCGGACATCTACGTTACGGACTCTACTACCGAAGCGAAAAAACTGTTCAACGATAAGATTTGGCCTGCGATCGATAAAATCCTGCAGCAAAACGCCGAAACCTGTCCGATCCTGTCTGAGAAATGGTCTGGTATTCACGTTAGCGGTGATCAGCTCAAGGGTCAGCGCCACAAACAGGAAGATCGCTTCCTGGCTTACCCTAACGGTCAATATATGGACCGTGGTGAGGACCCGATTTCTGTTCTGGCGGTTTTCGATGGTCACGGCGGTCACGAATGCTCTCAGTACGCGGCAGGTCATCTGTGGGAAACGTGGCTGGAGGTGCGTAAAAGCCGCGACCCGTCTGACTCTCTGGAGGACCAGCTGCGCAAATCTCTCGAACTGCTGGACGAACGTATGACCGTGCGTTCTGTCAAAGAATGCTGGAAAGGCGGTTCTACCGCTGTCTGCTGTGCAATCGACATGGACCAGAAGCTCATGGCGCTCGCATGGCTGGGCGACTCTCCGGGCTATGTTATGTCTAACATCGAATTCCGTCAACTCACTCGTGGTCATTCTCCTTCTGATGAGCGCGAAGCACGTCGCGTAGAGGAGGCAGGTGGCCAACTCTTTGTAATCGGTGGTGAACTCCGTGTTAACGGTGTGCTGAACCTGACGCGCGCACTCGGCGATGTTCCGGGTCGTCCGATGATCTCTAACGAACCGGAAACTTGCCAGGTTCCGATCGAATCTAGCGACTATCTCGTGCTCCTGGCGTGCGATGGTATCAGCGACGTATTCAATGAGCGTGACCTGTATCAACTGGTCGAAGCGTTTGCCAATGATTACCCGGTTGAAGACTACGCGGAACTGTCTCGCTTTATTTGCACCAAGGCCATTGAAGCAGGCTCTGCGGACAATGTATCTGTTGTTATTGGTTTCCTGCGTCCGCCGCAGGACGTGTGGAAACTGATGAAGCATGAATCTGACGACGAAGACTCTGATGTTACCGATGAGGAATAA
GC% Content for gene (codon optimized): 52.59%
Primer design results for 'tail' primers (this is just 2 sequences):
Forward Primer: (Underlined = pLIC Primer Attachment)
5’-- TACTTCCAATCCATGGAAAAAGTTAATGAAGAA --3’
Reverse Primer: (underlined = pLIC Reverse Primer Attachment)
5’-- TATCCACCTTTACTGTTATTCCTCATCGGTAACATC --3’